Hydrogen peroxide is a reaction product produced by many enzymatic reactions in which oxidase takes part. For example, reactions such as oxidation of glycerine by glycerine oxidase, oxidation of glucose by glucose oxidase, or oxidation of cholesterol by cholesterol oxidase, are very important for analyses, particularly in the area of medical diagnostics.
Known methods for measuring hydrogen peroxide which is enzymatically formed are based on a titrimetric method, potentiometry, polarography, quantitative colorimetry, and an enzymatic method using the enzyme catalase or peroxidase. In accordance with a method for enzymatic measurement using peroxidase, a chromogen which reacts with hydrogen peroxide in the presence of peroxidase to form a dye which can be measured by photometry is used as an indicator. These kinds of known reagents for measuring hydrogen peroxide include an indicator system described in P. Trinder, Ann. Clin. Biochem., Vol. 6 (1969), pages 24-27. In this system, measurement is carried out using photometry after phenol is allowed to oxidatively bond with the chromogen: 4-aminoantipyrine in the presence of peroxidase by the action of hydrogen peroxide. It is possible to use other phenol compounds such as 1,7-dihydroxynaphthalene instead of phenol.
When the compounds of the present invention are allowed to react with hydrogen peroxide in the presence of peroxidase and a known color former (for example, phenol, p-chlorophenol, 1,7-dihydroxynaphthalene, N,N-di-substituted m-toluidine, etc.), a dye is formed by an oxidative coupling reaction. Accordingly, if the resulting dye is quantitatively measured by colorimetry, the amount of hydrogen peroxide can be determined. Accordingly, if various oxidase type enzymes and their substrates which are already used for clinical examination are coexistent, instead of carrying out the direct action of hydrogen peroxide, the amount of hydrogen peroxide formed by an enzyme reaction can be determined. As a result, it is possible to determine the activity of these enzymes and the amount of the substrates (for example, glucose, uric acid, GPT (glutamyl-pyruvate transaminase), GOT (glutamyl-oxaloacetate transaminase), creatinine, etc.).
Conventionally, the quantitative analysis based on such a principle has been carried out by so-called wet quantitative analysis which is carried out in a solution in a test tube. Recently, so-called dry quantitative analysis using a multilayer analysis sheet having a reagent layer containing a reagent has been developed. The compounds of the present invention can be incorporated in the reagent layer of such a multilayer analysis sheet, by which good results are obtained.